The objective of this proposal is to define the role of innate and adaptive immune mechanisms in the development of insulin resistance and Type II diabetes mellitus (T2DM). T2DM and insulin resistance (IR) are diseases involving inflammation of the adipose and are typified by increases in macrophage (innate) and T cell infiltration of visceral adipose stores and a phenotypic change to a proinflammatory and CD3+ T cell (adaptive) accumulation. The relationship and the relative contributions of these components to T2DM and IR are poorly understood. We have recently observed a dramatic decrease in T regulatory cells (Tregs) in the visceral fat of DIO C57BL/6 mice compared to lean controls with no alteration in splenic populations. Tregs are characterized by the expression of the transcription factor FoxpS, have immunosuppressive functions, can be demonstrated in tissue niches and are protective in chronic inflammatory states, such as atherosclerosis. The long term objective of this project is to show a role for Tregs in the etiology of insulin resistance and the role of innate immune cells in influencing Treg function and differentiation. Our specific aims are: 1. Investigate the relationship between the development of insulin resistance in diet induced obesity (DIO) in mice and to extend these observations to human adipose samples. 2. Demonstrate that adipose tissue-derived macrophages (CD11b+ cells) and dendritic cells (CD11c+ cells) prevent Treg differentiation in-vitro and to elucidate the mechanism. 3. Determine the impact of systemic Treg depletion on the development of insulin resistance and adipose inflammation. We propose utilizing a unique model that allows us the ability to track and accurately quantify Tregs based upon expression of green fluorescent protein (Foxp3-GFP knockin mice, C57BL/6 background). DIO in these mice will be accomplished by high fat feeding. In-vivo experiments in conjunction with novel in-vitro co-culture experiments that will utilize macrophages and dendritic cells isolated from the adipose of obese and lean wild type mice, co-cultured with splenic CD3+CD4+CD25-GFP- T cells from Foxp3-GFP knockin mice. These experiments will further our understanding of how the innate and adaptive immune systems modulate inflammatory processes in the development of insulin resistance and T2DM in humans. Lay Summary: Insulin resistance and type II diabetes are a burgeoning health crisis in the US and around the world due to increasing rates of obesity. Understanding how the immune system, affected by obesity, promotes these metabolic conditions is central to understanding, preventing, and treating this complex metabolic condition.